This project will use cultured vertebrate neurons as model systems in which to study mechanisms of neurotoxicity, particularly developmental neurotoxicity. The developing nervous system is a sensitive target for neurotoxic substances, since the immature blood-brain barrier fails to exclude many toxicants, and relatively small changes in pathfinding or connectivity of individual neurons may lead to abnormal neuronal wiring patterns, which may in turn lead to behavioral changes, from subtle to overt. Although the mechanisms whereby neurotoxicants exert their effects are multiple and varied, our investigations will be based on three specific hypotheses: (1) A variety of neurotoxicants affect Ca2+ homeostasis by altering Ca2+ influx or extrusion through the plasma membrane, or by altering Ca2+ sequestration and/or release from intracellular stores. (2) Changes in transmembrane Ca2+ flux and/or intracellular free Ca2+ concentration ([Ca2+]in) play important roles in regulating neuronal development. (3) Therefore, a toxicant that significantly alters Ca2+ fluxes and/or [Ca2+]in, at least during critical periods, is likely to alter neuronal development. This project will investigate mechanisms of developmental neurotoxicity of several classes of neurotoxicants, specifically inorganic Pb2+, triethyl lead, lindane, permethrin, and cypermethrin. Our specific aims are to investigate (1) mechanism whereby neurotoxicants alter transmembrane Ca2+ fluxes and [Ca2+]in; (2) mechanisms whereby neurons may regulate Ca2+ fluxes and [Ca2+]in in the face of continued toxicant exposure; and (3) the relationship between toxicant-induced changes in Ca2+ fluxes and [Ca2+]in, and neurite development. We will use a variety of techniques including: (a) detailed morphometric analysis of neuronal differentiation in the presence of toxicants; (b) calcium imaging using fura-2 to measure [Ca2+]in during toxicant exposure, while manipulating the external and internal milieu, to determine the sources of Ca2+ that contribute to elevating [Ca2+]in; (c) voltage clamping of neurons to determine if toxicants affect voltage-sensitive ion channels in ways that could explain the observed alterations in [Ca2+]in; and (d) biochemical and biophysical analyses to assess toxicant effects on plasma membrane CaATPase and Na+-K+ATPase pumps and Na+-Ca2+ exchange proteins.